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Metastatic medullary thyroid cancer (MTC) is a rare but often aggressive thyroid malignancy with a 5-year survival rate of less than 40% and few effective therapeutic options. Adoptive T cell immunotherapy using chimeric antigen receptor (CAR)-modified T cells (CAR Ts) is showing encouraging results in the treatment of cancer, but development is challenged by the availability of suitable target antigens. We identified glial-derived neurotrophic factor (GDNF) family receptor alpha 4 (GFRα4) as a putative antigen target for CAR-based therapy of MTC. We show that GFRα4 is highly expressed in MTC, in parafollicular cells within the thyroid from which MTC originates, and in normal thymus. We isolated two single-chain variable fragments (scFvs) targeting GFRα4 isoforms a and b by antibody phage display. CARs bearing the CD3ζ and the CD137 costimulatory domains were constructed using these GFRα4-specific scFvs. GFRα4-specific CAR Ts trigger antigen-dependent cytotoxicity and cytokine production in vitro, and they are able to eliminate tumors derived from the MTC TT cell line in an immunodeficient mouse xenograft model of MTC. These data demonstrate the feasibility of targeting GFRα4 by CAR T and support this antigen as a promising target for adoptive T cell immunotherapy and other antibody-based therapies for MTC., Graphical Abstract, Metastatic medullary thyroid carcinoma (MTC) is a rare but aggressive malignancy with no curative options. By sequencing mRNA from a patient’s tumor, Siegel and colleagues identified and characterized GFRα4 as a promising tumor target antigen and demonstrated that CAR T immunotherapy targeting this antigen controlled MTC in tumor xenograft models.

While many decades of scientific research studies have gone into harnessing the power of the immune system to fight cancer, only recently have cancer immunotherapeutic approaches begun to show robust clinical responses in patients with a variety of cancers. These treatments are adding to the current arsenal of cancer treatments; surgery, radiation and chemotherapy, and increasing the therapeutic options for cancer patients. Despite these advances, issues associated with these therapies include that not all patients respond to these therapies, and some patients who respond experience varying degrees of toxicities. One of the major issues affecting immunotherapy is the inability to evaluate trafficking of activated T-cells into sites of tumor. The current diagnostic imaging based on conventional anatomic imaging, which is the mainstay to monitor response to cytotoxic chemotherapy or radiation, is not adequate to assess initial response to immunotherapy or disease evolution. Patients’ prognosis by histological analysis has limited use in regards to immunotherapy. Thus, there is a crucial need for noninvasive biomarkers for screening patients that show long term response to therapy. Here, we provide a brief account of emerging molecular magnetic resonance imaging biomarkers that have potential to exploit the metabolism and metabolic products of activated T cells.

152 Background: Glioblastoma (GBM) is a deadly cancer, with a 5-year survival rate of less than 10%. T cells can be redirected to kill cancer cells using chimeric antigen receptors (CAR), a promising method to treat solid tumors. IL13Rα2 is expressed in many solid tumors but not normal tissues, and provides a target for CAR T cells as a powerful immunotherapy treatment. Methods: Human and canine tumors were screened for IL13Rα2 expression by flow cytometry and RT-PCR. Different IL13Rα2 single-chain variable fragments (scFv) were tested in CARs in vitro against human and canine recombinant IL13Rα2 protein to identify a cross-reactive clone. IL13Rα2 CARs expressed in human and canine T cells and showed antigen-specific stimulation by cytokine secretion and target cell lysis. Solid tumors were used to establish in vivo subcutaneous and orthotopic xenograft models in NSG mice. Mice received a single treatment of 2-5 million CAR T cells IV with or without anti PD-1, CTLA-4 or TIM3 mAb checkpoint blockade. Results: IL13Rα2 was detected on three human GBM (D270, U251 and U87), four canine osteosarcomas (BW-, CS-, MC- and SK-KOSA) and two canine lung cancer cell lines (Cacal3, Cacal5). Two different IL13Rα2 CAR were generated that recognized human IL13Rα2 and not IL13Rα1, one also recognized canine IL13Rα2. Five million IL13Rα2 CAR T-cells delivered IV were able to eliminate established GBM tumors. Use of fewer CAR T cells initially controlled tumor growth, followed by tumor outgrowth correlated with expression of T cell exhaustion markers. Combined treatment with CARs plus checkpoint blockade mAb delivered IP had synergistic effects, restoring anergic T-cell function and improving tumor treatment. Conclusions: IL13Rα2 is highly expressed on human and canine tumors, but not normal tissues. IL13Rα2 specific CAR T cells successfully respond to human and canine tumors and treat GBM in a xenograft mouse model, although this effect was transient at lower treatment doses. Addition of checkpoint blockade mAbs to CAR therapy was beneficial for the treatment of solid tumors. We plan to utilize this treatment in a canine spontaneous cancer preclinical model and translate into clinical trials for patients with GBM.